PROJECT/SUMMARY ABSTRACT Ribosome biogenesis occurs in the nucleolus, a membrane-less organelle formed through liquid-liquid phase separation (LLPS). However, little is known on how phase separation influences ribosome biogenesis and how ribosomal RNA (rRNA) moves through the nucleolus. Recent work demonstrated rRNA and nucleophosmin (NPM1) undergo LLPS, but the molecular determinants driving this interaction are not known. My preliminary data indicates rRNA structure regulatesrRNA/NPM1 LLPS, suggesting a structure-based regulatory mechanism. To confirm this, I will perform experiments to probe changes in rRNA conformation upon stabilization of rRNA structure with Mg2+, and after LLPS with NPM1. I anticipate that NPM1 preferentially binds and stabilizes rRNA in a structure that promotes ribosomal protein (r-protein) association. To bind and co-fold with rRNA, r-proteins must enter the GC via a poorly understood mechanism, I propose that rRNA entry and its restructuring by NPM1 enhances r-protein entry. Once in the GC, r-protein/rRNA co-folding leads to abrogation of rRNA/NPM1 LLPS, allowing rRNA to be released as a pre-ribosomal particle into the nucleoplasm. To test this, I will determine r- protein partitioning into the GC and perform reconstitution studies using the E.Coli 30S small subunit. The studies proposed above will provide insights into how rRNA escapes the GC, but not how rRNA moves through the nucleolus. I propose the high rate of rDNA transcription leads to a flux of rRNA outward from the inner layer of the nucleolus via a mass action mechanism. I will develop an in vitro system to examine if the outward flux of rRNA and inward flux of r-proteins can recapitulate the early stages of ribosome biogenesis. In summary, the proposed studies will provide novel insights into the role of phase separation in ribosome biogenesis and provide the foundations for understanding how dysregulation of LLPS could lead to disease states such a Diamond- Blackfan Anemia (a type of ribosomeopathy), and mutant NPM1-associated leukemias (e.g., acute myeloid leukemia, acute promyelocytic leukemia, and anaplastic large cell lymphoma).